Epidemiological investigations have demonstrated that menopause is associated with an increased risk of CVD, showing that sex differences in the incidence and severity of CVD are no longer found after menopause.1,2⇓ Moreover, women who undergo early menopause, either surgically or naturally, are at increased risk of developing coronary heart disease in comparison with premenopausal women of the same age.3 Sex-hormone deficiency, independently of aging, seems therefore to be responsible for the enhanced risk of CVD, as also suggested by the favorable effects of ERT on cardiovascular morbidity and mortality.4,5⇓

Although there is increasing evidence of the protection afforded by ERT against CVD, the underlying mechanisms remain not fully explained. Estrogens are believed to play a protective role on the cardiovascular system mainly by a beneficial action on lipid profile, especially by favorably affecting plasma concentration of lipoproteins, by interfering with cholesterol deposition in the arterial wall and by preserving LDL from oxidation.6–8⇓⇓ Besides these lipid-mediated effects, recent data point to a direct vascular action of estrogens, which have been demonstrated to improve vascular reactivity, possibly by calcium channel blocking-like action9 and/or by interplaying with endothelial vasoactive factors.10,11⇓

It is now well documented that the endothelium plays a primary role in the modulation of vascular tone through the production of different factors, of which the most important is NO, a labile substance released by endothelial cells during the catabolism of l-arginine into citrulline through the activity of the NO synthase enzyme.12 There is clinical and experimental evidence suggesting that exogenous estrogens affect endothelium-dependent vasodilation of peripheral and coronary arteries in estrogen-deficient animals13–15⇓⇓ and in menopausal women.16–19⇓⇓⇓ Acute estrogen administration has been reported to improve vasoreactivity in healthy postmenopausal women at dosages achieving serum concentrations typical of the midcycle of reproductive-age women.20 Moreover, a beneficial effect of acute estrogen administration on myocardial ischemia has been found in postmenopausal women with angiographically proven coronary artery disease.21 On the other hand, endogenous estrogen seems to play a protective role on endothelial function since in normotensive women the age-related impairment of endothelium-dependent vasodilation occurs later in comparison with men, appearing only around the time of menopause.22,23⇓ Moreover, a protective effect of endogenous estrogen has also been shown in essential hypertensive women.22

Therefore, postmenopause is characterized by the loss of an estrogen-dependent mechanism protecting vascular function. However, no reports are yet available on the effect of acute endogenous estrogen deprivation on vasomotor response in women of reproductive age. Thus, this study was planned to assess the effect of surgical menopause on endothelium-dependent vasodilation in fertile women who underwent bilateral ovariectomy for uterine leiomyoma.

Methods

Subjects

A group of 10 women, aged 47.4±1.6 (range 45 to 51) years, with symptomatic uterine leiomyoma associated with menorrhagia (n=8), pelvic pain (n=1), or rapid increase in size (n=1) was enrolled in the study. All patients had regular menstrual cycles, no history of hypertension, CVD, dyslipidemia, diabetes mellitus, obesity, or any other major systemic illness. As control subjects, we enrolled 10 healthy women, aged 48.2±2.9 (range 43 to 50) years, who were matched for hemodynamic and humoral characteristics (Table 1). No patients had received hormone treatment or had had a pregnancy for at least 6 months before the study. All subjects had been free from any drugs for at least 4 weeks. Only 2 were mild smokers (less than 5 cigarettes a day).

Hemodynamic and Humoral Characteristics of Healthy Control Women and Patients Who Underwent Ovariectomy (mean±SEM)

In accordance with institutional guidelines, all patients were aware of the investigational nature of the study and gave written consent to it.

Experimental Procedure

All studies were performed at 8:00 am, after overnight fasting, with the subjects lying supine in a quiet air-conditioned room (22°C to 24°C). A polyethylene cannula (21 gauge, Abbot) was inserted into the brachial artery under local anesthesia (2% lidocaine). The cannula was connected through stopcocks to a pressure transducer (model MS20, Electromedics) for the determination of systemic mean arterial blood pressure (one third pulse pressure plus diastolic pressure) and heart rate (model VSM1, Physiocontrol), and for intra-arterial infusions by a Harvard infusion pump (series 944A, Ealing). FBF was measured in both forearms (experimental and contralateral) by strain-gauge venous plethysmography (LOOSCO, GL LOOS).24 The circulation to the hand was excluded 1 minute before each measurement of FBF by inflating a pediatric cuff around the wrist, at supra-systolic blood pressure. Earlier work has determined the sensitivity and the reproducibility of the method.25

Forearm volume was measured by the water-displacement method, and infusion rates of drugs were normalized to 100 mL of tissue by altering the drug concentration in the solvent, while the speed of infusion was not modified. The drugs used were infused through separate ports via three-way stopcocks at concentrations that had no systemic effects.

Experimental Design

Endothelium-dependent vasodilations were estimated by performing a dose-response curve to intra-arterial acetylcholine (cumulative increase of the infusion rates: 0.15, 0.45, 1.5, 4.5, 15 μg/100 mL forearm tissue per minute, for 5 minutes at each dose),26,27⇓ while endothelium-independent vasodilations were assessed with a dose-response curve to intra-arterial sodium nitro-prusside28 (cumulative increase by 1,2, and 4 μg/100 mL forearm tissue per minute, for 5 minutes each). These rates were selected to induce vasodilation comparable to that obtained with acetylcholine. The sequence of infusion of the two drugs was randomized and 45 minutes of recovery were allowed between the two experimental steps.

Forearm study was performed during the follicular phase in both control subjects and patients. Patients repeated the study within 1 month after ovariectomy (24±2 days). Finally, 6 of 10 patients were studied again 3 months after ERT (transdermal estradiol; TTS 50, 50 μg/24 h).

Data Analysis

Data were analyzed in terms of changes in FBF. Since mean arterial blood pressure did not significantly change during the study, increments in FBF were taken as evidence of local vasodilation. Results were expressed as mean±SD in text and as mean±SEM in the figures and tables. Data were analyzed statistically by the t test for paired or unpaired observations and by ANOVA for repeated measures. Scheffé’s test was applied for multiple comparison testing. A value of P<.05 was considered to be statistically significant.

Drugs

Acetylcholine HCl (Farmigea SpA) and sodium nitroprusside (Malesci) were obtained from commercially available sources and diluted freshly to the desired concentration by adding normal saline. Sodium nitroprusside was dissolved in glucosate solution and protected from light by an aluminum foil.

Results

The systemic demographic, hemodynamic, and humoral characteristics for control subjects and patients are summarized in Table 1. There was no significant difference in baseline values between control subjects and patients. Moreover, after ovariectomy and ERT, there was no statistically significant modification in basal parameters compared with values found before surgery. It is worth noting that no statistical difference in blood pressure values or lipid and glucose profile was observed when basal conditions, ovariectomy, and ERT were compared. Moreover, plasma estradiol values did not differ between control subjects and patients (76.5±34.3 and 71.6±31.3 pg/mL, respectively) and as expected fell to undetectable levels after ovariectomy. Finally, during ERT, plasma estrogen levels were 48.6±13.9 pg/mL.

Response to Intrabrachial Acetylcholine and Sodium Nitroprusside

Vasodilation to acetylcholine was not significantly different between control subjects (FBF rose from 3.6±0.4 to a maximum of 25.1±4.9 mL/100 mL forearm tissue per minute with the highest dose) and patients before surgery (FBF rose from 3.5±0.5 to a maximum of 23.4±4.5 mL/ 100 mL forearm tissue per minute with the highest dose) (Fig 1). The vasodilating effect of the endothelium independent vasodilator sodium nitroprusside was also similar in control subjects and patients (FBF rose from 3.4±0.4 to a maximum of 22.3±3.6 mL/100 mL forearm tissue per minute with the highest dose and from 3.4±0.4 to a maximum of 21.3±3.1 mL/100 mL forearm tissue per minute, respectively; NS) (Fig 1).

After ovariectomy the response to acetylcholine was significantly (P<.001) blunted compared with preintervention results (maximum to acetylcholine: from 3.4± 0.4 to 13.9±2.8 mL/100 mL forearm tissue per minute) (Fig 2) while the vasodilation to sodium nitroprusside was not altered (maximum to sodium nitroprusside: from 3.5±0.4 to 19.4±3.7 mL/100 mL forearm tissue per minute) (Fig 2).

Fig 2. FBF increase above basal (b) induced by intra-arterial acetylcholine (micrograms per 100 mL forearm tissue per minute) (left) and sodium nitroprusside (micrograms per 100 mL forearm tissue per minute) (right) in female patients with uterine leiomyoma (n=10) before (•) and after (○) ovariectomy. Data are shown as mean±SEM and expressed as absolute values. Asterisks denote a significant difference between patients before and after ovariectomy or after ERT (P<.05).

Finally, the effect of ERT was evaluated in a subgroup of our study population (6 of 10) (Table 2). In these subjects, vasodilation to acetylcholine, which before ovariectomy was similar to values observed in control subjects (maximum to acetylcholine: from 3.6±0.4 to 25.8±3.9 mL/100 mL forearm tissue per minute), was found to be significantly blunted after surgery (maximum to acetylcholine: from 3.3±0.4 to 14.7±2.4 mL/100 mL forearm tissue per minute; P<.05 versus preovariectomy) (Fig 3). However, after 3 months of ERT the vasodilating effect of acetylcholine was significantly (P<.02) increased compared with postovariectomy results (maximum to acetylcholine: from 3.5±0.5 to 25.5±3.1 mL/100 mL forearm tissue per minute) (Fig 3) and it is worth noting that it was no longer statistically different compared with preovariectomy values (Fig 3). In contrast, in the same subgroup of patients, the response to sodium nitroprusside (maximum to sodium nitroprusside: from 3.4±0.4 to 22.4±3.1 mL/100 mL forearm tissue per minute) observed before ovariectomy was unchanged either after surgery (maximum to sodium nitroprusside: from 3.1± 0.4 to 19.2±2.8 mL/100 mL forearm tissue per minute; NS versus preovariectomy) or ERT (maximum to sodium nitroprusside: from 3.4±0.4 to 23.6±3.2 mL/100 mL forearm tissue per minute; NS versus preovariectomy or postovariectomy) (Fig 3).

Hemodynamic and Humoral Characteristics of the Subgroup of Patients (n=6) Who Underwent Ovariectomy and 3 Months of ERT (mean±SEM)

Discussion

The present study was designed to test the effect of acute endogenous estrogen deprivation on endothelium-dependent vasodilation in women in reproductive age. Therefore, in a group of female patients who underwent bilateral ovariectomy for uterine leiomyoma, we evaluated the forearm vascular response to acetylcholine and sodium nitroprusside, an endothelium-dependent and -independent vasodilator, respectively. As control subjects we selected healthy women well matched with the study group population for demographic and clinical parameters. Particular attention was paid to risk factors impairing endothelial function such as age, blood pressure, phase of the menstrual cycle, lipid and glucose profile, and smoking history. In the two study populations, the response to acetylcholine and sodium nitroprusside was similar, indicating preserved endothelium-dependent vasodilation in patients with leiomyoma. In contrast, after bilateral ovariectomy, the response to acetylcholine was found to be reduced compared with preintervention results. Since the vasodilating effect of sodium nitroprusside remained unchanged, this finding suggests that acute estrogen deprivation leads to selective impairment of endothelium-dependent vasodilation. This possibility is further confirmed by results obtained after ERT. Thus, in a subgroup of ovariectomized patients (6 of 10), ERT significantly increased the response to acetylcholine compared with that observed after ovariectomy. Moreover, the effect of acetylcholine after ERT was no longer different compared with the response observed in basal conditions (before ovariectomy) or in the control group. Since again the vasodilating effect of sodium nitroprusside did not change, these findings not only indicate that ERT restores endothelium-dependent vasodilation in short term estrogen deprived women but also further confirm the positive effect of estrogen on endothelial function.

The most likely explanation for the beneficial effect of estrogen on endothelium-dependent vasodilation, as observed in our experimental conditions, is represented by a direct effect of the hormone on endothelial function. Although the exact mechanism by which endogenous estrogen protects the endothelium cannot be determined from the present study, experimental evidence indicates that administration of estrogen upregulates the transcription of NO synthase, the key enzyme regulating NO production.29 In addition, a further possible explanation is suggested by the finding that estrogen has antioxidant properties and may preserve endothelium-dependent vasodilation through the negative effect of superoxide anions that strongly inactivate NO.30

An alternative explanation may be represented by an indirect estrogen effect mediated by lipid profile changes associated with sex hormone deprivation or replacement. Estrogen may directly improve lipid profile by decreasing plasma concentrations of total and LDL cholesterol while increasing high-density lipoprotein cholesterol, and inhibiting oxidation of the LDL fraction.6,8⇓ Since both plasma lipid concentration and oxidative LDL status may directly impair endothelial function, the possibility exists that an estrogen-mediated beneficial effect on these parameters could improve endothelium-dependent vasodilation. While the possibility of an estrogen effect on lipid plasma concentration is very unlikely in our experimental conditions, since the short periods of estrogen deprivation and replacement (1 and 3 months, respectively) do not induce significant and clinically relevant changes in the lipid profile (see Table 1), a possible reduction of LDL oxidation cannot be ruled out by the present study. A final indirect possibility may be an estrogen-mediated effect on blood pressure values or glucose profile. How-ever, the present findings indicating that these parameters did not significantly change throughout the study seem to be sufficient to exclude this possibility.

The possibility of a direct effect of sex hormones on endothelial function, as suggested by the impairment in vasodilating response after acute estrogen deprivation, is in keeping with the finding of increased endothelium-dependent vasodilation after acute infusion of estradiol in cynomolgus monkeys15 and in postmenopausal women.18,20,21⇓⇓ Moreover, physiological changes in endogenous estrogen during the menstrual cycle have recently been found to correlate positively with flow-mediated vasodilation in the absence of lipid modifications, accounting for a direct action of estradiol in modulating endothelial function.31

Only few reports are available so far on the effect of chronic estrogen administration on the endothelium of ovariectomized experimental animals and postmenopausal women undergoing replacement therapy. An improvement in endothelium-dependent vasodilation after chronic estrogen administration has been found in atherosclerotic coronary arteries of ovariectomized monkeys14 and in both the brachial artery16 and epicardial coronary arteries17–19⇓⇓ of postmenopausal women.16,19⇓ In contrast, a recent report from Gilligan et al32 failed to demonstrate an increased vasodilating response to acetylcholine in postmenopausal women after 3 weeks of ERT by transdermal estradiol administered at an even higher dose as in our present investigation. Although this study seems to be at variance with our results, both the different population characteristics, mainly age and time elapsed since menopause and the different length of ERT could account for the different findings. Thus, increasing age is associated with a decline in endothelium-dependent vasodilation,33,34⇓ and this alteration is amplified in postmenopausal women.22 Therefore, it is important to observe that we studied younger women shortly after ovariectomy-induced menopause, while Gilligan et al evaluated an older study population, in most of whom menopause occurred years before. Furthermore some evidence suggests that estrogen receptor levels within the vasculature may be closely related to the hormonal milieu and may require a period of estrogen priming.35,36⇓ Therefore, both the number of months or years since menopause and a more lasting exogenous estrogen exposure may be crucial in regulating receptor exposure to exogenous estrogen administration. The difference in age and duration of menopause of the present study population compared with that evaluated by Gilligan et al32 and the different period of treatment can thus be taken into account to explain the discrepancy in the results.

Endothelial dysfunction has been demonstrated in pathological conditions to be associated with an increased risk of CVD.33,34,37–40⇓⇓⇓⇓⇓ The pivotal role of endothelium in the pathogenesis of vascular disease has been well documented by elucidating its contribution to control of the interaction between blood and the vessel wall.41 The impaired endothelial responses associated with the ovarian estrogen loss may contribute to explaining the rise in incidence and severity of CVD in women who have early surgical menopause. Thus, there is evidence suggesting that the demonstrated endothelial dysfunction at forearm level may reflect a more diffuse vascular alteration, as indicated by a report showing a close relationship between brachial artery flow-mediated vasodilation and endothelial changes in coronary arteries.42

Our study, performed roughly 1 month after ovariectomy, allows the hypothesis of a direct effect of sex hormone deprivation on vascular reactivity, occurring before lipid pattern changes, and provides further evidence of an estrogen action on endothelial function by the findings of a positive effect of 3-month ERT.

In conclusion, these findings indicate that endothelium-dependent vasodilation in women is inlhrenced by hormonal status, which positively modulates endothelial function, suggesting that these mechanisms may be at least partially responsible for the protective role of estrogen in the development of CVD in women.